1. Field of the Invention
The present invention relates to a microchip for analysis which is used for an analysis of biomolecular interactions or the like, an analysis system having the same, and an analysis method.
2. Description of the Related Art
Conventionally, in order to analyze a biomolecular interaction such as protein or the like, an analysis method in which, for example, an analyte is bound to a ligand and the state of binding reaction (for example, binding strength, binding rate, etc.) is detected using a phenomenon of surface plasmon resonance (SPR) is known. In particular, analysis apparatuses as described in Non-Patent Documents 1 and 2 have been widely used.
An example of such an analysis method will be described in summary. A ligand is preliminarily modified on a base layer (metal film) of a reaction bath section, a sample solution containing an analyte is supplied to the reaction bath section with a predetermined amount at a predetermined rate to allow a binding reaction between the ligand and the analyte, and light is irradiated onto the base layer of the reaction bath section, which results in surface plasmon resonance. Further, since a dielectric constant of the base layer changes according to the binding reaction between the ligand and the analyte and there occurs a phase shift in the resonance signal, the relationship between the reaction time and the state of binding can be determined by measuring the phase shift. In addition, based on the above-described relationship, identification of specific intermolecular binding, screening of substances that are unknown of whether they undergo binding, ranking of the binding strength among molecules, quantification of analyte concentration, calculation of dissociation constant, calculation of kinetics constant or the like can be performed.
FIG. 16 schematically shows such an analysis apparatus. In the analysis apparatus, a sample solution supply channel 103 to which a sample solution vessel 101A for storing sample solution containing analyte is connected via a pump 102A is connected to a reaction bath section 105, in which a binding reaction is performed, via a large-volume fixed-quantity storage section 104. In particular, the fixed-quantity storage section 104 and the reaction bath section 105 are schematically shown. Valves 106A and 106B are respectively disposed upstream and downstream of the fixed-quantity storage section 104; Further, the reaction bath section 105 is connected to a waste solution tank 107. On the other hand, a buffer solution supply channel 108 to which a buffer solution vessel 101B for storing buffer solution is connected via a pump 102B is connected between the valve 106A of the sample solution supply channel 103 and the fixed-quantity storage section 104 via a valve 106C. Then, a waste solution channel 109 which is linked to the waste solution tank 107 is connected between the fixed-quantity storage section 104 and the valve 106B downstream thereof via a valve 106D.
An analysis method using the analysis apparatus will be specifically described with reference to a flowchart in FIG. 17. First, a ligand is preliminarily modified in the reaction bath section 105 (Step 201). Then, the extraction of air is performed to discharge air in each flow channel. For example, the extraction of air from the buffer solution supply channel 108, the fixed-quantity storage section 104, and the reaction bath section 105 is performed by closing the valves 106A and 106D, opening the valves 106C and 106B, and operating the pump 102B to flow the buffer solution from the buffer solution vessel 101B to the waste solution tank 107 via the buffer solution supply channel 108, the fixed-quantity storage section 104, and the reaction bath section 105 (Step 202). Subsequently, the-extraction of air from the sample solution supply channel 103, the fixed-quantity storage section 104, and the waste solution channel 109 is performed by closing the valves 106C and 106B, opening the valves 106A and 106D, and operating the pump 102A to flow the sample solution from the sample solution vessel 101A to the waste solution tank 107 via the sample solution supply channel 103, the fixed-quantity storage section 104, and the waste solution channel 109 (Step 203). In such a manner, the extraction of air from the flow channels is completed. Further, at the time when a predetermined quantity (for example, 50 μl) of sample solution remains in the fixed-quantity storage section 104, the valve 106A is closed and the valve 106C is opened. Then, the valve 106D is closed, the valve 106B is opened, and the pump 102B is operated to transfer the buffer solution to the buffer solution supply channel 108. Thus, the sample solution in the fixed-quantity storage section 104 is purged therefrom by the buffer solution (Step 204). When the purged sample solution passes through the reaction bath section 105, the ligand modified in the reaction bath section 105 undergoes a binding reaction with the analyte in the sample solution. Then, by measuring a resonance signal resulting at that time by an optical method or the like, the state of reaction is detected (Step 205). Further, the quantity of the sample solution used in the analysis is 50 μl as stored in the fixed-quantity storage section 104, the reaction time is, for example, from 5 to 50 minutes, and the flux of the sample solution by the operations of the pumps 106A and 106B is from 1 to 10 μl/min.
FIG. 18 shows an example of measuring the resonance signal when the binding reaction is actually performed by the above-described analysis method. In this example, when the sample solution begins to flow in the reaction bath section 105 while the buffer solution is still flowing in the reaction bath section 105, the change in phase of the resonance signal increases, thus indicating the initiation of the binding between the analyte and the ligand. However, after a while, equilibrium in concentration is reached, and binding does not occur between analyte and ligand so much, such that the change in phase of the resonance signal is stopped. Then, when the supply of a predetermined quantity of sample solution is completed and when the buffer solution is supplied again to the reaction bath section 105, a part of the bound analyte-ligand undergoes dissociation, thereby decreasing the change in phase of the resonance signal. The detection of the dissociation state is effective in, for example, knowing the binding strength between the analyte and the ligand. Thereafter, though not shown in FIGS. 16 and 17, a regeneration solution, instead of the buffer solution, is supplied to the reaction bath section 105 to purge all analyte by dissociating it from the ligand and the ligand in the reaction bath section 105 is brought into a reusable state.
The reaction bath section 105 in the above-described analysis apparatus according to a related art has a configuration in which a window 100a is provided on a substrate 100 and the window 100a is covered by a sensor chip 110 (see FIGS. 19 and 20). Further, the ligand is modified in the sensor chip 110 which serves as a lid. In this configuration, the reaction state is examined by irradiating a light beam from an optical means (not shown in FIG. 18) outside the substrate 100 to be reflected. That is, since the binding reaction between the ligand and the analyte causes a change in the dielectric constant and thus in the refractive index of light in the base layer, the detection of the reflection of incident light can give information on the state of binding reaction. Further, if the reaction bath section 105 where the reaction and the analysis are performed has a configuration in which the window 100a is opened on the substrate 100, as shown in FIG. 19, the vertical spacing d in the reaction bath section 105 is larger than other parts, and thus it is difficult to obtain a desired spacing (for example, 50 μm). In addition, there may be stagnation in the flow of a fluid around a corner portion 105a. Thus, it is impossible to obtain a uniform flow and there is possibility that a complete extraction of air is not achieved, with residual air still remaining. Therefore, as shown in FIG. 20, a so-called “flow cell” morphology can be employed so as to reduce the spacing d in the reaction bath section 105, such that a uniform flow can be achieved without stagnation. At the same time, the analyte in the sample solution can be allowed to flow around the ligand-modified sensor chip 110 so as to increase the probability for the analyte to come into contact with the ligand, thus leading to an efficient binding reaction.
[Patent Document 1] US Patent Application Publication No. 2002/0128593
[Non-Patent Document 1] Kaori Morimoto, “A plasmon resonance analysis type analysis apparatus,” Clinical Examination (Igaku-Shoin, Ltd.), October 2003, Vol. 47, No. 11, Special Issue for 2003, p. 1319-1327
[Non-Patent Document 2] Kazuhiro Nagata and Hiroshi Handa, “Real-Time Analysis of Biomolecular Interactions,” Springer-Verlag, Tokyo, November 1998
According to the above-described analysis method of the related art, the predetermined quantity (for example, 50 μl) of the sample solution is stored in the fixed-quantity storage section 104 and the sample solution is supplied to the reaction bath section 105 by purging with the buffer solution. In this case, since the fixed-quantity storage section 104 is required to be of a large capacity, it is difficult to form each flow channel of the analysis apparatus on a minute microchip and a relatively large substrate 100 is needed, thereby enlarging the analysis apparatus as a whole. Further, since the substrate 100 is large and expensive and the sample solution vessel 101A and the pump 102A, and the buffer solution vessel 101B and the pump 102B are connected to the sample solution supply channel 103 and the buffer solution supply channel 108 on the substrate 100, respectively, mounting or removal is not easy. Thus, in consideration of the complexity of the operation and the increase in the product cost, it has been difficult in practice to replace the substrate 100 with a disposable one.
Since it has been difficult so far in practice to use and discard the substrate 100, the substrate 10 has been used repeatedly in a variety of analyses. In that case, even though the flow channels or the pumps are cleaned with a cleaning solution, there still is a risk that the analyte in the sample solution of the previous analysis remains somehow in the flow channels or the pumps to possibly react with the sample solution for the subsequent analysis, thereby lowering reliability of analysis. Further, in the case of supplying the sample solution to the reaction bath section 105 by purging with the buffer solution, there is a risk that both solutions may diffuse into each other at an interface, thus making the point of completion of the reaction unclear, or making the concentration of the sample solution inaccurate, to impair reliability of the analysis result. In addition, it is difficult in practice to use and discard the substrate 100 and the substrate 100 should be used repeatedly in a number of analyses. Thus, as described above, a configuration in which a part of the reaction bath section 105 is constituted by a separable sensor chip 110 and the sensor chip 110 is modified with the ligand is adopted. Accordingly, the above-described flow cell structure is generally used (see FIG. 20).